Tetanus toxin elicits an electrophysiologic response in primary neuronal cultures similar to that observed in the nervous system in situ. A biochemical assay for neurotransmitter release demonstrated that toxin-induced convulsant activity is related to a reduction in the synaptic release of inhibitory neurotransmitters and a concomitant two-fold increase in the release of excitatory neurotransmitters. These patterns of release parallel increases and decreases in the frequency of recorded excitatory and inhibitory postsynaptic potentials. The time-course of toxin action could be modified by increasing and reducing neuronal activity during toxin exposure, indicating that the level of endogenous activity may be a factor in differential toxin action. Immunohistochemistry for acidic compartments revealed that tetanus toxin collapses the neuronal endosomal pH gradient much the same as monensin, and that monensin interferes with the ability of the toxin to act at the synaptic terminal. Thus, the toxin molecule appears to enter the neuron through receptor-mediated endocytosis and to require an acid pH in the endosome in order to gain entry into the neuronal cytoplasm.